title: Equine CRISP3 modulates interaction between spermatozoa and polymorphonuclear neutrophils.
abstract: Equine spermatozoa induce a uterine inflammatory response characterized by a rapid, transient influx of polymorphonuclear neutrophils (PMNs). Seminal plasma proteins have been shown to modulate the interaction between spermatozoa and PMNs, but a specific protein responsible for this function has not been identified. The objective of this study was to isolate and identify a protein in equine seminal plasma that suppresses binding between spermatozoa and PMNs. Seminal plasma was pooled from five stallions, and proteins were precipitated in 60% (w/v) ammonium sulfate and dialyzed (3500 MW cutoff). Proteins were submitted to a Sephacryl S200 column, and fractions were pooled based on the fraction pattern. Each pool was analyzed for protein concentration and tested for its suppressive effect on PMN/sperm binding. Protein pools with biological activity were submitted to ion-exchange chromatography (diethylaminoethyl [DEAE] Sephadex column) with equilibration buffers containing 0.1-0.5M NaCl. Eluants were pooled, analyzed for protein concentration, and tested for suppressive effects on PMN/sperm binding. Protein distribution and purity were determined by one- and two-dimensional SDS-PAGE, and the purified protein was submitted for sequence analysis and identification. This protein was identified as equine CRISP3 and was confirmed by Western blotting. Suppression of PMN/sperm binding by CRISP3 and seminal plasma was confirmed by flow cytometry (22.08% ± 3.05% vs. 2.06% ± 2.02% vs. 63.09% ± 8.67 for equine seminal plasma, CRISP3, and media, respectively; P < 0.0001). It was concluded that CRISP3 in seminal plasma suppresses PMNs/sperm binding, suggesting that CRISP3 regulates sperm elimination from the female reproductive tract.
authors.affiliation: Department of Animal Sciences, College of Agriculture and Life Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA.
authors.forename: A
authors.initials: A
authors.lastname: Doty
authors.forename: W C
authors.initials: WC
authors.lastname: Buhi
authors.forename: S
authors.initials: S
authors.lastname: Benson
authors.forename: K E
authors.initials: KE
authors.lastname: Scoggin
authors.forename: M
authors.initials: M
authors.lastname: Pozor
authors.forename: M
authors.initials: M
authors.lastname: Macpherson
authors.forename: M
authors.initials: M
authors.lastname: Mutz
authors.forename: M H T
authors.initials: MH
authors.lastname: Troedsson
mesh_terms: D000595:Amino Acid Sequence; D000645:Ammonium Sulfate; D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005434:Flow Cytometry; D006736:Horses / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism*; D029589:Seminal Plasma Proteins / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D013094:Spermatozoa / Q000378:metabolism*
chemical_list: D029589:Seminal Plasma Proteins; D000645:Ammonium Sulfate